PROJECT SUMMARY The small molecule products of the cultured microbes have served as a major source of leads compounds for antibiotics discovery. However, novel antibiotics are needed to combat antibiotics resistance, and a continued focus on the abundant molecules from the cultured microbes is ineffective due to high rates of rediscovery. Here we propose computational techniques for discovering novel small molecules from environmental and host-oriented microbiomes. Advances in 16S and shotgun metagenomics have revolutionized our understanding about the microbial composition of various communities and their biosynthetic gene clusters (the sets of genes that synthesize microbial small molecules from simple building blocks). Preliminary genome-mining results from our group and others show that environmental and host-associated metagenomes contain thousands of biosynthetic gene clusters with uncharacterized small molecule products. Moreover, our results show that the tandem mass spectrometry data collected on these microbiomes harbor the signals for many known and novel small molecules, making them an untapped gold mine for future antibiotics discovery. The overarching aim of this proposal is to develop computational and probabilistic approaches for integrating shotgun metagenomics and tandem mass spectrometry data from multiple microbiome sources to discover the small molecule products of the biosynthetic gene clusters. We will do this by (i) detecting the pairs of co-occurring molecular features and microbial producers by a comparative analysis of the abundances of molecules and microbial species across multiple microbial communities, (ii) connecting the pair of molecular features and microbial species to the putative biosynthetic gene clusters by metagenome mining (iii) connecting the triple from the previous step to the predicted molecular structures of the biosynthetic gene clusters, and (iv) scoring predicted molecular structures against the corresponding mass spectra, and identifying the matches with high statistical confidence. We will apply these methods to discover novel antimicrobial small molecules from the publicly available shotgun metagenomics and tandem mass spectrometry datasets collected from the environmental and host-associated microbiomes including Earth Microbiome Project and American Gut Project. The tools developed in this proposal will be accessible to the community through the Global Natural Product Social molecular networking platform.